Mutations in the HBV PreS/S gene related to hepatocellular carcinoma in Vietnamese chronic HBV-infected patients.

BACKGROUND: Chronic hepatitis B virus (CHB) infection is a major health problem and leading cause of hepatocellular carcinoma (HCC) worldwide. Several point and deletion mutations on the PreS/S gene have been intensively considered associated with HCC. This study aimed to describe the characteristics of HBV PreS/S mutations in Vietnamese CHB-infected patients and their association with HCC.
METHODS: This cross-sectional study was conducted from 02/2020 to 03/2021, recruited Vietnamese CHB-infected patients with HBV-DNA >3 log10-copies/mL and successful PreS/S gene sequencing. Mutations were detected by direct Sanger sequencing.
RESULTS: 247 CHB-infected patients were recruited, characterized by 68.8% males, 54.7% HBV genotype B, 57.5% HBeAg positive, 23.1% fibrosis score ≥F3 and 19.8% HCC. 61.8% amino acid replacements were detected throughout the PreS1/PreS2/S genes. The most common point-mutations included N/H51Y/T/S/Q/P (30.4%), V68T/S/I (44.9%), T/N87S/T/P (46.2%) on PreS1 gene; T125S/N/P (30.8%), I150T (42.5%) on PreS2 gene; S53L (37.7%), A184V/G (39.3%), S210K/N/R/S (39.3%) on S gene. The rates of case(s) with any point-mutation on the Major Hydrophylic Region (MHR) and the "a" determinant region were 63.6% and 39.7%, respectively. Most of S point-mutations were presented with low rates such as T47A/E/V/K (9.3%), P120S/T (8.5%), G145R (2%). On multivariable analysis, males (OR = 4.51, 95%CI 1.78-11.4, p = 0.001), age≥40 (OR = 5.5, 95%CI 2.06-14.68, p = 0.001), W4P/R/Y on PreS1 (OR = 11.56, 95%CI 1.99-67.05, p = 0.006) and 4 S point-mutations as: T47A/E/V/K (OR = 3.67, 95%CI 1.19-11.29, p = 0.023), P120S/T (OR = 3.38, 95%CI 1.09-10.49, p = 0.035), S174N (OR = 29.73, 95%CI 2.12-417.07, p = 0.012), P203R (OR = 8.45, 95%CI 1.43-50.06, p = 0.019) were associated with HCC.
CONCLUSIONS: We detected 61% amino acid changes on PreS/S regions in Vietnamese CHB patients. One point-mutation at amino acid 4 on PreS1 gene and 4 point-mutations at amino acids 47, 120, 174, and 203 on S gene were associated with HCC. Further investigations are recommended to further clarify the relationship and interaction between mutations in HBV genome and HCC progression.

Introduction

Chronic HBV (CHB) infection affects 296 million people worldwide in 2019 [1], and has been considered as a major global health problem. CHB infection is the leading cause of liver cirrhosis and contributes over 50% of hepatocellular carcinoma (HCC) [2]. Vietnam locates in the HBV-high-prevalence area in Asia [3] which has the high incidence of HBV related-HCC [2]. It was reported that 62.3% of HCC cases and 81.3% of advanced HCC cases in Vietnam were HBV infected [4, 5].

HBV belongs to the Hepadnaviridae family with an incomplete double strand DNA genome, which carries 4 overlapped open reading frames, coding for 4 proteins PreS/S, preCore/Core, Pol, and X. HBsAg proteins are trancsripted from PreS/S open reading frame that consists of 3 surface proteins: Small (S), Medium (M) and Large (L). The S protein that drives the releasing of viral particles consists of 227 amino acids (aa). The M protein that enriches the secretion of virion contains an extra N-terminal extension of 55 aa. The L protein that is involved in the interaction with core particles in the packaging of virion at the endoplasmic reticulum (ER) has a further N-terminal extension of 108 or 119 aa–depending on genotypes [6]. In the absence of L protein, S protein is secreted alone as noninfectious subviral particles. L protein can suppress the subviral particle secretion depending on the L/S protein ratio. During natural HBV infection subviral particles outnumber virions by a factor of 1000:1. The principal epitopes of HBsAg mainly locate in the “a” determinant (aa 124–147) in the major hydrophilic region (MHR) induces the neutralized B cell responses.

Mutations in PreS/S gene result in deletion of surface proteins or synthesis of varieties of truncated proteins. Especially, mutations on the C-terminal region (aa 179–226) of S gene contribute in retention of HBsAg within the hepatocytes ER [7, 8], activate multiple oncogenic signal pathways, promote the growth of hepatocytes and eventually lead to HCC development. Multiple scientific evidences had proved PreS mutations as prediction markers for HCC development and recurrence of HBV-related HCC [9-12]. The relationship between PreS/S gene mutations and HCC were studied distinctly on the PreS region [13]. Mutations at T53C, PreS deletions, PreS2 start codon, C7A, A2962G, C2964A and C3116T in the PreS region have been proved that significantly increase risk of HCC [14, 15]. Wang et al. (2006) had concluded that the retention of L antigens from the PreS mutants caused ER stress, induced oxidative DNA damage, and resulted in genomic instability. The L antigens from the PreS mutants are attributed to the upregulation cyclooxygenase-2 and cyclin A, and promotion of cell cycle and hepatocytes proliferation [16].

Mutations on the S gene in Vietnamese CHB patients had been described in the earliest study since 2012. Dunford et al. (n = 187) had reported a rate of 31% cases with point-mutation in the immunodominant ‘a’ region, especially two major vaccine escape mutations with minor rates as G145A/R (2.2%) and P120L/Q/S/T (5.3%) had been detected [17]. The mutation of PreS deletion with a rate of 20% was reported from Matsuo et al. (2017) in Vietnamese CHB-infected patients [18]. Bui TTT et al. (2017) described more concretely about point-mutations (N38E 71.9%, N38K 71.1%, A60V/E 100% on the PreS1 region, L126T/S 77% on the PreS2 and N3S 27.4% on the S region) [19]. In a multicenter study on 660 patients from China, Korea and Vietnam, Kim et al. (2018) [20] had reported 237 amino acid mutations in the MHR on the S region. There was not any report on mutations and their association with HCC on the PreS/S gene in Vietnamese patients.

In this study, we described the characteristics of HBV PreS/S gene mutations in Vietnamese patient with CHB and analyzed the relations of these mutations with HCC.

Materials and methods

Study design and population

The cross-sectional study had been conducted at the Hepatology Clinic of University Medical Center (UMC), Ho Chi Minh city from February 2020 to March 2021. There were 293 male and female CHB patients paticipated in this study, who met the inclusion criteria of being older than 18 years old, had HBsAg positive more than 6 months, no previous nucleos(t)ide analogues treatment (NAs) and HBV DNA >3 log10-copies/mL. Their serum samples were extracted from 4 mL blood, stored in -80 celcius degree for PreS/S gene sequencing. Serum samples of 247 patients that had been successfuly sequenced the HBV PreS/S gene were analyzed for the final results. Among them, there were 212 CHB patients whose serum samples had been stored during 2014–2016 and 35 CHB patients were recruited in 2020–2021.

Variables and measurements

Personal characteristics, times from diagnosis of CHB infection and HBV markers were collected from the hospital electronic database. HCC was defined for cases with tumor detected on abdominal ultrasound, serum alpha-fetoprotein (AFP) >20 ng/mL and was confirmed on abdominal CT scan with focal lesions with early arterial phase enhancement and rapid "washout" in venous phase [21]. Cirrhosis was defined as having signs of portal hypertension (splenomegaly, ascites, vascular collaterals on abdominal wall, esophageal varices or portal hypertensive gastropathy on gastroscopy) and signs of insufficiency of liver function (palmar erythema, vascular spiders, low concentration of albumin (<35 g/dL), elevation of the international normalized ratio (INR >1.1), thrombocytopenia (<160,000/mm3)) as well as irregularity of hepatic surface on ultrasonography or F3 on Metavir score using Acoustic radiation force impulse (ARFI) measurement [22].

HBsAg quantification (Elecsys HBsAgII Quant-Roche kit), HBeAg (Cobas-Roche kit) and HBV DNA quantification (using the AccuPid HBV Quantification kit (KT-Biotech)) with limits of detection ≥300 copies/mL, linear range: 300 to 108 copies/mL) were performed at the University Medical Center laboratory. HBV genotype (B or C) was determined based on the sequence of S gene.

PreS/S mutation analyzed by sanger sequencing

PreS/S mutations were analyzed at Center for Molecular Biomedicine of University of Medicine and Pharmacy (UMP) at Ho Chi Minh City. HBV DNA was extracted from serum using the GeneJetTM Viral DNA and RNA Purification kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Two sets of overlapping primers were then used to amplify the whole PreS/S region of HBV with TaKaRa TaqTM HotStart Polymerase (Takara Bio, Shiga, Japan). Primers for the PreS1/PreS2 region were: FA2-L (5’- TTGAGAGAAGTCCACCACGAG-3’) and FA2-R (5’-GCGTCGCAGAAGATCTCAAT-3’); S region were FA3-L (5’-CTGCTGGTGGCTCCAGTT-3’); FA3-R (5’-GCCTTGTAAGTTGGCGAGAA-3’). PCR involved initial denaturation at 98°C for 3 min followed by 45 cycles of 98°C for 10 sec, 58°C for 30 sec, and 72°C for 60 sec, with a final elongation of 72°C for 2 min. PCR products were checked for size and purity using 1.5% agarose gel electrophoresis. PCR product was purified enzymatically using the ExoSAP-ITTM PCR Product Cleanup Reagent (Thermo Scientific, Waltham, MA) to remove excess primers and dNTPs before Sanger sequencing using the BigDye Terminator v3.1 Kit and the ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). PCR fragment was sequenced and analyzed in both directions. The sequences were compared to the reference sequence of genotype B (GenBank_AB073846) and genotype C (GenBank_X04615) using the CLC Main Workbench software (Qiagen, Germany).

Statistics

The SPSS 25.0 software was used to analyze the data. Percentage was used to present the rates of point-mutations, the rates of possessing at least one mutation and the rates of genes deletion on each region. The Chi-square test (or Fisher exact test) was used to compare the distributions of mutations among groups with or without HCC. Multivariable analysis with logistic regression was used to find out factors related to HCC. Two-side p value of <0.05 was considered statistically significant.

Ethics considerations

The study was done based on the Declaration of Helsinki. Stored serum samples and all variables included in the study was approved by the Ethics Committee of the University of Medicine and Pharmacy at Ho Chi Minh City (Reference number: 119/HDDD). Informed consents were obtained from all participants prior to the study.

Results

Characteristics of the study population

The study included 247 CHB patients, 68.8% were males, 57.9% were older than 40 years of age and older. 57.5% were positive with HBeAg marker, 83% had HBV DNA ≥5 log10 copies/mL, 54.7% genotype B. 23.1% were with liver fibrosis and 19.8% were with HCC ().

Characteristics of the detected mutations on the PreS1, PreS2 and S genes in overall population and the HCC subgroups

There were 248/401 (61.8%) amino acid replacements that were detected throughout the PreS1/PreS2/S genes on the overall populations. In the PreS1 region, 57.1% replacements (68/119) were found. The mutations with a rate over 30% were N51Y/T/S/Q/P 30.4%, V68T/S/I 44.9%, and T/N87S/T/P 46.2%; from 10 to <30% were Q10K/H/R (16.2%), H48Y/N (13.8%), E/D54A/N (25.1%), I/N56H/W (25.9%), K57Q/K (25.1%), A60V (25.5%), D/A62S/T (25.1%), G73S/N (24.3%), and V95A (24.3%); and from 5 to <10% were G35R/K (8.5%), and I84V/M/L (7.7%). These above mutations were not differently distributed among non-HCC and HCC group. Interestingly, most of amino acid changes (54/68) in the preS1 were presented with the rates <5%. Among them, 4 point-mutations W4P/R/Y, S5L/T, A90T/S/G, and L108V/I were observed with the significantly higher rates in the HCC group compared to non-HCC group: W4P/R/Y (12.2% vs 2%, p = 0.005), S5L/T (6.1% vs 1%, p = 0.055), A90T/S/G (6.1% vs 0.5%, p = 0.026) and L108V/I (6.1% vs 0.5%, p = 0.026) (

In the PreS2 region, 41/55 (74.5%) amino acid changes were detected. The point-mutations with the rates of over 30% were T125S/N/P (30.8%), I150T (42.5%), and V158A (36%); from 10 to <30% of population were M120V/I (11.3%) and F141V/L/I (11.3%); and from 5 to <10% of population were Q121R/K (5.3%), T164I/D/S (6.1%), and F165S/L (5.7%). As same as the PreS1 region, most of amino acid changes on the PreS2 region (34/41 sites) were presented at a rate <5% (Table 3). Only F141V/L/I was found with a significantly higher rate in the HCC group (18.4% vs 9.6%, p = 0.08).

Table 3

Distribution of point and deletion mutations on the PreS2 gene (n = 247).

PreS2 (aa 120–174)	Overall population n (%)	HCC n (%)			p a
		Yes (n = 49)	No (n = 198)
Point-mutations
M120V/I	28 (11.3)	7 (14.3)		21 (10.6)	0.47
Q121R/K	13 (5.3)	1 (2.0)		12 (6.1)	0.47
W122R	3 (1.2)	1 (2.0)		2 (1.0)	0.49
N123T/K	3 (1.2)	1 (2.0)		2 (1.0)	0.49
S124T	8 (3.2)	1 (2.0)		7 (3.5)	0.29
T125S/N/P	76 (30.8)	12 (24.5)		64 (32.3)	0.29
T126N/I/A	3 (1.2)	1 (2.0)		2 (1.0)	0.49
H128L	4 (1.6)	1 (2.0)		3 (1.5)	1
Q129K	1 (0.4)	1 (2.0)		0	0.2
A130T/N	12 (4.9)	3 (6.1)		9 (4.5)	0.71
L/Q132I/H	5 (2.0)	1 (2)		4 (2)	1
D133N	1 (0.4)	0		1 (0.5)	1
P134H/T	2 (0.8)	0		2 (1.0)	1
R135K	2 (0.8)	0		2 (1.0)	1
V136A	1 (0.4)	1 (2.0)		0	0.2
R137K/Q	4 (1.6)	1 (2.0)		3 (1.5)	1
A138D	1 (0.4)	1 (2.0)		0	0.2
L139Q/P	2 (0.8)	0		2 (1.0)	1
Y140S/N/H/F/C	9 (3.6)	2 (4.1)		7 (3.5)	1
F141V/L/I	28 (11.3)	9 (18.4)		19 (9.6)	0.08
A143V	1 (0.4)	0		1 (0.5)	1
S146F	2 (0.8)	0		2 (1.0)	1
S147G	1 (0.4)	0		1 (0.5)	1
S148L	4 (1.6)	0		4 (2.0)	0.59
G149E/K	7 (2.8)	2 (4.1)		5 (2.3)	0.63
I150T	105 (42.5)	16 (32.7)		89 (44.9)	0.12
S152N	1 (0.4)	1 (2.0)		0	0.2
P153L	1 (0.4)	0		1 (0.5)	1
Q155P/H	3 (1.2)	2 (4.1)		1 (0.5)	0.1
N156T/I/S	7 (2.8)	1 (2.0)		6 (3.0)	1
T157S	2 (0.8)	0		2 (1.0)	1
V158A	89 (36.0)	18 (36.7)		71 (35.9)	0.91
A160T/P	5 (2.0)	1 (2.0)		4 (2.0)	1
I161T/L	4 (1.6)	0		4 (2.0)	0.59
T164I/D/S	15 (6.1)	3 (6.1)		12 (6.1)	1
F165S/L	14 (5.7)	3 (6.1)		11 (5.6)	1
K167T	11 (4.5)	2 (4.1)		9 (4.5)	1
T168I	4 (1.6)	0		4 (2.0)	0.59
V172A	3 (1.2)	1 (2.0)		2 (1.0)	0.49
P173Q/L	4 (1.6)	1 (2.0)		3 (1.5)	1
N174S/K	2 (0.8)	1 (2.0)		1 (0.5)	0.36
PreS2 deletion	40 (16.2)	11 (22.4)		29 (14.6)	0.18

percentage by column, a Chi-square test.

The PreS1 deletion was detected in 27.5% (68 patients), and the PreS2 deletion was observed in 16.2% (40 patients) (Tables

In the S region, 61.2% amino acid changes were detected (139/227). The rate of cases with at least one point-mutation detected on the "a” determinant region (aa 124–148) was 39.7% and on the MHR region (aa 100–160) was 63.6%. The HCC group had significant higher rate of possessing ≥1 point-mutation on the MHR region (79.6% vs 59.6%, p = 0.009).

The point-mutations on the S gene that owned the rates of >30% of the population were: S53L (37.7%), A184V/G (39.3%), and S210K/N/R/S (39.3%); from 15 to <30% were L21S (29.1%), G44E/V (18.6%), I126T/N/S (21.1%), and M198I/M (18.2%); and from 5 to <15% were V14A/G/Q (10.1%), N40S/K (6.9%), T47A/E/V/K (9.3%), P/L49R/H (5.7%), P62Q/L (9.7%), C76Y/T/W (10.5%), Y100C/F (5.3%), P120S/T (8.5%), R122K (8.9%), M133T/S/L/I (7.7%), Y161F/S (10.1%), T189I (5.3%), S204R/N (10.1%), I208T/S (5.7%), L213I/M (7.3%), and V224A (11.7%). Half of the amino acid changes (116/227) on the S region had the detection rates <5% and most of them were not related to HCC. Exceptionally, 13 S mutations presented the higher distributions (p<0.1) in the HCC group: F20S (8.2% vs 1%, p = 0.015), D33G (4.1% vs 0%, p = 0.039), (T47A/E/V/K (18.4% vs 7.1%, p = 0.025), R79H (6.1% vs 0%, p = 0.007), L88P (4.1% vs 0%, p = 0.039), P120S/T (on the MHR region, 16.3% vs 6.6%, p = 0.042), G145R (6.1% vs 1%, p = 0.055), S174N (6.1% vs 0.5%, p = 0.026), V190A (6.1% vs 0.5%, p = 0.026), P203R (8.2% vs 2%, p = 0.052), Y206H/F/C (6.1% vs 1.5%, p = 0.094), L209V/S/G (6.1% vs 1%, p = 0.055) and F212Y/L/C (8.2% vs 0.5%, p = 0.006), ().

The point-mutations on the Pres1/Pres2/S genes related to HCC–multivariable analysis

Nineteen point-mutations that distributed differently (p<0.1) among the HCC and non-HCC groups were checked to remove their interactions using multivariable analysis (Tables 2–4). Six point-mutations remained related to HCC. They were one mutation on the PreS1 region: W4P/R/Y (OR = 5.48, 95%CI 1.32–22.83) and 5 mutations on the S region: F20S (OR = 9.72, 95%CI 1.55–61.06), T47A/E/V/K (OR = 2.91, 95%CI 1.04–8.13), P120S/T (OR = 4.26, 95%CI 1.58–11.52), S174N (OR = 18.21, 95%CI 1.77–187.65), and P203R (OR = 9.72, 95%CI 1.55–61.06) ().

Table 2

Distribution of point and deletion mutations on the PreS1 gene (n = 247).

PreS1 (aa 1–119)	Overall population n (%)	HCC n (%)		pa
		Yes (n = 49)	No (n = 198)
Point-mutations
G2R/G	1 (0.4)	0	1 (0.5)	1
W4P/R/Y	10 (4)	6 (12.2)	4 (2.0)	0.005 b
S5L/T	5 (2)	3 (6.1)	2 (1.0)	0.055 b
S6A	3 (1.2)	1 (2.0)	2 (1.0)	0.49
K7N	1 (0.4)	0	1 (0.5)	1
P8T	1 (0.4)	1 (2.0)	0	0.2
Q10K/H/R	40 (16.2)	8 (16.3)	32 (16.2)	0.98
T14I/T	1 (0.4)	0	1 (0.5)	1
S17F/A	4 (1.6)	1 (2.0)	3 (1.5)	1
P19S	1 (0.4)	0	1 (0.5)	1
F25L	1 (0.4)	1 (2.0)	0	0.2
D27G/S	33 (13.4)	6 (12.2)	27 (13.6)	0.8
I31T	1 (0.4)	1 (2.0)	0	0.2
P32L	3 (1.2)	0	3 (1.5)	1
A33F/L	2 (0.8)	1 (2.0)	1 (0.5)	0.36
F34Y	1 (0.4)	0	1 (0.5)	1
G35R/K	21 (8.5)	3 (6.1)	18 (9.1)	0.78
S38T	1 (0.4)	0	1 (0.5)	1
N/E39K/G/D/A	11 (4.5)	2 (4.1)	9 (4.5)	1
N40Y/T	2 (0.8)	0	2 (1.0)	1
D42N	1 (0.4)	0	1 (0.5)	1
D44H/N	3 (1.2)	0	3 (1.5)	1
L45R/F	10 (4.0)	3 (6.1)	7 (3.5)	0.42
H48Y/N	34 (13.8)	3 (6.1)	31 (15.7)	0.08
D50N/E	2 (0.8)	1 (2.0)	1 (0.5)	0.36
N/H51Y/T/S/Q/P	75 (30.4)	15 (30.6)	60 (30.3)	0.97
E/D54A/N	62 (25.1)	9 (18.4)	53 (26.8)	0.23
A55S	1 (0.4)	0	1 (0.5)	1
I/N56H/W	64 (25.9)	14 (18.6)	50 (25.3)	0.64
K57Q/K	62 (25.1)	13 (26.5)	49 (24.7)	0.80
G59A	1 (0.4)	1 (2.0)	0	0.2
A60V	63 (25.5)	14 (28.6)	49 (24.7)	0.58
D/A62S/T	62 (25.1)	15 (30.6)	47 (23.7)	0.32
P65T	1 (0.4)	0	1 (0.5)	1
F67V/L	2 (0.8)	0	2 (1.0)	1
V68T/S/I	111 (44.9)	16 (32.7)	95 (48)	0.053 a
P70S	1 (0.4)	0	1 (0.5)	1
G73S/N	60 (24.3)	11 (22.4)	49 (24.7)	0.74
L74M/V	6 (2.4)	1 (2.0)	5 (2.5)	1
L75V/M	5 (2.0)	0	5 (2.5)	0.59
W77R/G	5 (2.0)	1 (2.0)	4 (2.0)	1
S78N	3 (1.2)	1 (2.0)	2 (1.0)	0.49
Q80L/H	3 (1.2)	1 (2.0)	2 (1.0)	0.49
A81T	3 (1.2)	1 (2.0)	2 (1.0)	0.49
Q82L	1 (0.4)	0	1 (0.5)	1
I84V/M/L	19 (7.7)	3 (6.1)	16 (8.1)	0.77
L85I/H/F	3 (1.2)	1 (2.0)	2 (1.0)	0.49
T86A/S	6 (2.4)	2 (4.1)	4 (2.0)	0.34
T/N87S/T/P	114 (46.2)	18 (36.7)	96 (48.5)	0.14
V88I/M/L	5 (2)	2 (4.1)	3 (1.5)	0.26
P89R	1 (0.4)	0	1 (0.5)	1
A90T/S/G	4 (1.6)	3 (6.1)	1 (0.5)	0.026 b
A91T/P	8 (3.2)	1 (2.0)	7 (3.5)	1
P92L	1 (0.4)	0	1 (0.5)	1
P94T/S	5 (2.0)	2 (4.1)	3 (1.5)	0.26
V95A	60 (24.3)	12 (24.5)	48 (24.2)	0.97
T97I/T/A	4 (1.6)	0	4 (2.0)	0.59
N98T/K/I	3 (1.2)	0	3 (1.5)	1
S101T/L	2 (0.8)	0	2 (1.0)	1
G102R/K	2 (0.8)	0	2 (1.0)	1
R103T	1 (0.4)	0	1 (0.5)	1
Q104R/K	4 (1.6)	2 (4.1)	2 (1.0)	0.18
L108V/I	4 (1.6)	3 (6.1)	1 (0.5)	0.026 b
S109T	11 (4.5)	2 (4.1)	9 (4.5)	1
R113T	1 (0.4)	0	1 (0.5)	1
D114E	1 (0.4)	0	1 (0.5)	1
T115S/C	6 (2.4)	0	6 (3.0)	0.6
Q118L	1 (0.4)	0	1 (0.5)	1
A119V	1 (0/4)	0	1 (0.5)	1
PreS1 deletion	68 (27.5%)	15 (30.6)	53 (26.8)	0.59

percentage by column, a Chi-square test, b Fisher Exact test.

Table 4

Distribution of point-mutations on the S gene (n = 247).

S (aa 1–227)	Overall population n (%)	HCC n (%)		pa
		Yes (n = 49)	No (n = 198)
Point-mutations
E2G	1 (0.4)	0	1 (0.5)	1
N3S	1 (0.4)	0	1 (0.5)	1
T4I	1 (0.4)	0	1 (0.5)	1
A5T/S	11 (4.5)	2 (4.1)	9 (4.5)	1
F8P	1 (0.4)	0	1 (0.5)	1
L9P	3 (1.2)	1 (2)	2 (1)	0.49
G10R	2 (0.8)	1 (2.0)	1 (0.5)	0.36
P11H	1 (0.4)	0	1 (0.5)	1
L13P/V	2 (0.8)	0	2 (1.0)	1
V14A/G/Q	25 (10.1)	5 (10.2)	20 (10.1)	0.98
L15S	4 (1.6)	1 (2)	3 (1.5)	1
Q16P	2 (0.8)	0	2 (1.0)	1
A17E	2 (0.8)	0	2 (1.0)	1
G18R	2 (0.8)	1 (2.0)	1 (0.5)	0.36
F19S	1 (0.4)	1 (2)	0	0.20
F20S	6 (2.4)	4 (8.2)	2 (1.0)	0.015 b
L21S	72 (29.1)	13 (26.5)	59 (29.8)	0.65
L22W	1 (0.4)	0	1 (0.5)	1
R24K	4 (1.6)	0	4 (2.0)	0.59
I25V/A	4 (1.6)	0	4 (2.0)	0.59
T27I	2 (0.8)	0	2 (1.0)	1
I28T	1 (0.4)	0	1 (0.5)	1
Q30R/K	5 (2.0)	2 (4.1)	3 (1.5)	0.26
S31R	1 (0.4)	1 (2)	0	0.20
L32P	1 (0.4)	0	1 (0.5)	1
D33G	2 (0.8)	2 (4.1)	0	0.039 b
S34L	1 (0.4)	1 (2)	0	0.20
W35STOP	1 (0.4)	0	1 (0.5)	1
N40S/K	17 (6.9)	2 (4.1)	15 (7.6)	0.54
F41S	2 (0.8)	0	2 (1.0)	1
L42P/R	4 (1.6)	1 (2)	3 (1.5)	1
G43K	1 (0.4)	0	1 (0.5)	1
G44E/V	46 (18.6)	13 (26.5)	33 (16.7)	0.11
A45T/G/V	8 (3.2)	3 (6.1)	5 (2.5)	0.20
P46H/L	4 (1.6)	1 (2)	3 (1.5)	1
T47A/E/V/K	23 (9.3)	9 (18.4)	14 (7.1)	0.025 b
C48S	2 (0.8)	1 (2)	1 (0.5)	0.36
P/L49R/H	14 (5.7)	2 (4.1)	12 (6.1)	0.74
Q51L	2 (0.8)	0	2 (1.0)	1
S53L	93 (37.7)	20 (40.8)	73 (36.9)	0.61
S59N	1 (0.4)	0	1 (0.5)	1
S61L	10 (4.0)	2 (4.1)	8 (4.0)	1
P62Q/L	24 (9.7)	6 (12.2)	18 (9.1)	0.51
C64Y	1 (0.4)	0	1 (0.5)	1
P67Q	6 (2.4)	1 (2)	5 (2.5)	1
I68T	9 (3.6)	2 (4.1)	7 (3.5)	1
R73H	1 (0.4)	0	1 (0.5)	1
W74S/L	6 (2.4)	0	6 (3.0)	0.60
M75T	1 (0.4)	1 (2)	0	0.20
C76Y/T/W	26 (10.5)	7 (14.3)	19 (9.6)	0.34
L77R	7 (2.8)	3 (6.1)	4 (2.0)	0.14
R79H	3 (1.2)	3 (6.1)	0	0.007 b
F80S	1 (0.4)	0	1 (0.5)	1
F83S/C	3 (1.2)	0	3 (1.5)	1
C85Y/F	3 (1.2)	2 (4.1)	1 (0.5	0.1
L88P	2 (0.8)	2 (4.1)	0	0.039 b
L89P	1 (0.4)	0	1 (0.5)	1
I92T	4 (1.6)	0	4 (2.0)	0.59
F93S/C	4 (1.6)	1 (2)	3 (1.5)	1
L95W	5 (2.0)	1 (2)	4 (2.0)	1
V96G	1 (0.4)	1 (2)	0	0.20
L98V	2 (0.8)	1 (2.0)	1 (0.5)	0.36
Y100C/F	13 (5.3)	2 (4.1)	11 (5.6)	1
Q101K/H/R	12 (4.9)	2 (4.1)	10 (5.1)	1
M103T	2 (0.8)	0	2 (1.0)	1
L104W	1 (0.4)	0	1 (0.5)	1
I110L/Q	9 (3.6)	2 (4.1)	7 (3.5)	1
R112K	1 (0.4)	0	1 (0.5)	1
T113N	1 (0.4)	0	1 (0.5)	1
S114T/P/A	7 (2.8)	3 (6.1)	4 (2.0)	0.14
T115N	1 (0.4)	0	1 (0.5)	1
T116N	1 (0.4)	1 (2)	0	0.20
T118M	1 (0.4)	0	1 (0.5)	1
P120S/T	21 (8.5)	8 (16.3)	13 (6.6)	0.042 b
R122K	22 (8.9)	5 (10.2)	17 (8.6)	0.72
T123A/N	4 (1.6)	1 (2)	3 (1.5)	1
I126T/N/S	52 (21.1)	13 (26.5)	39 (19.7)	0.29
P127T/A/S	12 (4.9)	3 (6.1)	9 (4.5)	0.71
A128V	2 (0.8)	1 (2)	1 (0.5)	0.36
Q129R/N/L/H	5 (2.0)	2 (4.1)	3 (1.5)	0.26
G130R	1 (0.4)	0	1 (0.5)	1
T131N/S	10 (4.0)	2 (4.1)	8 (4.)	1
S132P	2 (0.8)	1 (2)	1 (0.5)	0.36
M133T/S/L/I	19 (7.7)	5 (10.2)	14 (7.1)	0.55
F134Y/V/L/I	6 (2.4)	2 (4.1)	4 (2.0)	0.34
S136F	1 (0.4)	1 (2)	0	0.20
T140I	7 (2.8)	1 (2)	6 (3.0)	1
T143M	3 (1.2)	0	3 (1.5)	1
D144E/A/D	2 (0.8)	0	2 (1)	1
G145R/A	5 (2.0)	3 (6.1)	2 (1)	0.055 b
N147S	1 (0.4)	1 (2)	0	0.20
P151H	1 (0.4)	0	1 (0.5)	1
W156L	2 (0.8)	1 (2)	1 (0.5)	0.36
A159V	9 (3.6)	3 (6.1)	6 (3.0)	0.39
R160K	6 (2.4)	2 (4.1)	4 (2.0)	0.34
Y161F/S	25 (10.1)	4 (8.2)	21 (10.6)	0.79
F162Y	1 (0.4)	0	1 (0.5)	1
Y163F	2 (0.8)	0	2 (1)	1
E164G	1 (0.4)	1 (2.0)	1 (0.5)	0.36
A166V/G	6 (2.4)	1 (2.0)	5 (2.5)	1
S167L	1 (0.4)	0	1 (0.5)	1
V168A	1 (0.4)	0	1 (0.5)	1
F170S	1 (0.4)	1 (2)	0	0.20
W172C	1 (0.4)	1 (2)	0	0.20
L173P	3 (1.2)	2 (4.1)	1 (0.5)	0.10
S174N	4 (1.6)	3 (6.1)	1 (0.5)	0.026 b
L175S	1 (0.4)	0	1 (0.5)	1
V177L	2 (0.8)	0	2 (1)	1
V180A	2 (0.8)	1 (2.0)	1 (0.5)	0.36
W182STOP	1 (0.4)	0	1 (0.5)	1
A184V/G	97 (39.3)	23 (46.9)	74 (37.4)	0.22
L186H	2 (0.8)	0	2 (1)	1
T189I	13 (5.3)	2 (4.1)	11 (5.6)	1
V190A	4 (1.6)	3 (6.1)	1 (0.5)	0.026 b
S193L	4 (1.6)	0	4 (2.0)	0.59
I195T	1 (0.4)	1 (2)	0	0.20
M198I/M	45 (18.2)	7 (14.3)	38 (19.2)	0.43
W199L/STOP	2 (0.8)	1 (2)	1 (0.5)	0.36
Y200F/W	12 (4.1)	2 (4.1)	10 (5.1)	1
P203R	8 (3.2)	4 (8.2)	4 (2.0)	0.052 b
S204R/N	25 (10.1)	7 (14.3)	18 (9.1)	0.28
L205V	1 (0.4)	1 (2)	0	0.20
Y206H/F/C	6 (2.4)	3 (6.1)	3 (1.5)	0.094 b
N207S	1 (0.4)	1 (2)	0	0.20
I208T/S	14 (5.7)	3 (6.1)	11 (5.6)	1
L209V/S/G	5 (2.0)	3 (6.1)	2 (1.0)	0.055 b
S210K/N/R/S	97 (39.3)	22 (44.9)	75 (37.9)	0.42
P211R	1 (0.4)	0	1 (0.5)	1
F212Y/L/C	5 (2.0)	4 (8.2)	1 (0.5)	0.006 b
L213I/M	18 (7.3)	5 (10.2)	13 (6.6)	0.37
L216STOP/Y	5 (2.0)	2 (4.1)	3 (1.5)	0.26
P217S/L	2 (0.8)	1 (2)	1 (0.5)	0.36
I218L	1 (0.4)	0	1 (0.5)	1
F220Y/L/C	7 (2.8)	0	7 (3.5)	0.35
C221Y/R	11 (4.5)	1 (2)	10 (5.1)	0.70
L222P	1 (0.4)	0	1 (0.5)	1
V224A	29 (11.7)	7 (14.3)	22 (11.1)	0.62
Y225F	1 (0.4)	1 (2)	0	0.20
I226M/T/S	5 (2.0)	0	5 (2.5)	0.59
S Functional sequence (≥1 point-mutations)
MHR	157 (63.6)	39 (79.6)	118 (59.6)	0.009
“a” determinant	98 (39.7)	24 (49.0)	74 (37.4)	0.14

percentage by column, a Chi-square test, b Fisher exact test.

Personal and viral characteristics were also evaluated for their confounding effects on the correlation between mutations and HCC. Five basal characters that had different distributions in HCC and non-HCC groups were male (83.7% vs 65.2%, p = 0.012), age group >40 (85.7% vs 51%, p<0.001), HBeAg negative (61.2% vs 37.9%, p = 0.003), HBV DNA <5 log10-copies/mL (46.9% vs 9.6%, p<0.001), and liver fibrosis of >F3 (38.8% vs 19.2%, p = 0.004) (.

The final multivariable analysis for related factors to HCC had included gender, age group, HBeAg marker and 6 mutations (PreS1 W4P/R/Y and five S point-mutations as F20S, T47A/E/V/K, P120S/T, S174N, P203R) on Table 5. The result found eight variables that composed of males (OR = 4.51, 95%CI 1.78–11.4, p = 0.001), age ≥40 (OR = 5.5, 95%CI 2.06–14.68, p = 0.001), HBeAg negative (OR = 2.46, 95%CI 1.1–5.53, p = 0.029) and 5 point-mutations such as W4P/R/Y (OR = 11.56, 95%CI 1.99–67.05, p = 0.006), T47A/E/V/K (OR = 3.67, 95%CI 1.19–11.29, p = 0.023), P120S/T (OR = 3.38, 95%CI 1.09–10.49, p = 0.035), S174N (OR = 29.73, 95%CI 2.12–417.07, p = 0.012) and P203S (OR = 8.45, 95%CI 1.43–50.06, p = 0.019) had significant relations with HCC (Table 6). S174N (S) had the highest OR (29.73) with positive predictive value (PPV), negative predictive value (NPV), sentitivity (SEN), specificity (SPE) for predicting HCC respectively, 75%, 81.1%, 6.1%, 99.5%. The predictive values of all other 4 point-mutations related to HCC were concretely described in , based on our current available data.

Table 5

Point-mutations related to HCC–multivariable analysis (n = 247).

Mutation	OR (95%CI)	p
W4P/R/Y (PreS1)	5.48 (1.32–22.83)	0.019
F20S (S)	9.72 (1.55–61.06)	0.015
T47A/E/V/K (S)	2.91 (1.04–8.13)	0.042
P120S/T (S)	4.26 (1.58–11.52)	0.004
S174N (S)	18.21 (1.77–187.65)	0.015
P203R (S)	9.72 (1.55–61.06)	0.016

The characteristics of these 6 point-mutations had been analysed and found that W4P/R/Y ( (p = 0.022) and T47A/E/V/K ( (p<0.001) had significant higher rates on genotype C; P120S/T ( had higher rates on genotype B (p<0.001), HBeAg (-) group (p = 0.019) and low HBV DNA group (<5 log10-copies/mL) (p = 0.013) (

Table 6

Multivariable analysis for related factors to HCC (n = 247).

Variables	OR (95%CI)	P
Sex (male)	4.51 (1.78–11.4)	0.001
Age group (≥40)	5.5 (2.06–14.68)	0.001
HBeAg negative	2.46 (1.1–5.53)	0.029
W4P/R/Y (PreS1)	11.56 (1.99–67.05)	0.006
T47A/E/V/K (S)	3.67 (1.19–11.29)	0.023
P120S/T (S)	3.38 (1.09–10.49)	0.035
S174N (S)	29.73 (2.12–417.07)	0.012
P203R (S)	8.45 (1.43–50.06)	0.019

Discussion

To the best of our knowledge, this investigation was one of the first studies on PreS/S gene mutations and their relation with HCC in Vietnamese CHB infected patients. The study population included CHB infected patients with HBV DNA >3 log10-copies/mL (for the higher chance of mutation detection) and had successful PreS/S sequencing (for better mutation description and analysis its correlation with HCC). The rate of mutations that were presented in this study therefore might be higher than that of the real HBV infected population in Vietnam. Our study sample composed of 54.7% genotype B, 57.5% HBeAg positive, 23.1% liver fibrosis of >F3, 83% HBV DNA >5 log10-copies/mL and especially 19.8% HCC accompanied. These special characteristics on the population were not only presented the variables that need to be adjusted for their confounding effects but also ensured the aim of detection of mutations and its relationships with HCC.

There were 61.8% amino acid replacements that were detected on the entire PreS/S gene. The rates of changes that were higher on the PreS2 and S gene (74.5% of 228 and 70% of 55 amino acid sites, respectively, versus 56.7% of 120 amino acid sites on the PreS1) revealed the high variability of these regions.

On the PreS1 region that consists of 119 amino acid, 57.1% amino acid replacements were detected with a wide range of mutation rates from 0.4% to 46.9%. However, 79.4% of these replacements (54/68) presented in less than 5% of population. These frequently observed point-mutations were mostly not related to HCC. Contrarily, 4 point-mutations that belong to the low-rate group were related to HCC. They were W4P/R/Y and S5L/T (p = 0.055) on the NTCP region; A90T/S/G on the HSP70 region and L108V/I on the S promoter and B cell epitopes (Table 2).

On the PreS2 region that consists of 55 amino acids, 74.5% amino acid changes were detected with the mutation rates ranged from 0.4% to 42.5%. 82.9% amino acid changes (34/41) belong to the group of <5% rates. Only F141V/L/I had the higher distribution in the HCC group (18.4% vs 9.6%, p = 0.08) (Table 3). Our finding seemed compatible with a report from Mun et al., who had found that F141L mutation strain increased the risk of HCC in HBV genotype C infected subjects [23]. They had also proved the enhanced cell cycling effects of F141L-expression cell lines through the doubling frequencies of colony-forming versus the wild types.

The PreS1 deletion (27.5%) and PreS2 deletion (16.2%) were equally distributed in the HCC and non-HCC groups (Table 4). The same rates of PreS deletion (20%) were reported from Matsuo et al. (2017) (on 5/21 Vietnamese CHB patients) [18] and from Choi et al. in Korean genotype C patients [24]. Literature reviews found mutations of these PreS genes effect on retaining of HBV inside the host’s cells and on malignant transforming of hepatic cells afterwards [7, 25–27]. More studies had specified on PreS1 deletion and HCC correlation (Zhang et al. (2017) [28], Choi et al. (2019) [29]).

On the S region, 61.2% amino acid changes were detected with the mutation rates range from 0.4% to 39.3%. We found mutations as Y100C/F, P120S/T, R122K, I126T/N/S, S132P, M133T/S/L/I, G145R (the vaccine escape mutant, 2%), Y200F/W and Y206H/F/C as same as that were reported from other studies (Hudu et al. (2015) [30], Kim et al. (2018) [20], Hazawa et al. (2018) [31], but amino acid change which had been described at amino acid 125 had not been detected in our study.

Moreover from our study, we often found the lower rates of amino acid changes compared to other studies such as from Bui TTT et al. (2017) (N38E 71.9%, N38K 71.1%, A60V/E 100% on the PreS1 region, L126T/S 77% on the PreS2 and N3S 27.4% on the S region) [19], from Kim et al. (2018) (K122R 69.3% on the S region compared to 8.9% R122K in our study) [20]. Inversely, we detected higher rates of S point-mutations as L21S (29.1%), S53L (37.7%), A184V/G (39.3%), S204R/N (10%) and S210K/N/R/S (39.3%), and also on the “a” determinant (39.7% cases with mutation, compared to 7% from Hudu’s group [30]. In spite of these lower and higher amino acid change rates, all of these mutations were found not related to HCC in our cross-sectional study. These differences in rates among studies could not only be explained by the distribution of genotype and by the varieties of subgroups in the study populations (such as the co-existence of HBsAg-AntiHBs status, nucleot(s)ide or immunoglobulin treatment, liver cirrhosis and HCC). Moreover, among our study population, antiHBs that had been tested on 186 cases with clinical symptoms were tested antiHBs and had detected 37 cases (19.9%) with HBsAg-AntiHBs co-exsistence, higher than the rates of 3–5% in other investigation [32, 33]. Therefore, it was presumed that CHB patients with varieties of presentations had been included in our study and contributed to the difference in rates of point-mutations compared to other studies.

We also found a significant higher distribution of cases with mutation on the MHR region (p = 0.009) in the HCC group, especially a higher rate of P120S/T. Outside of the MHR region we also detected higher rates of other 3 S mutations T47A/E/V/K, S174N (in the HLA II region) and P203S (in the HLA II region, the C-terminal domain) in the HCC group (Table 4). Hossini et al. (2019) had previously found the higher rate of P120T/S in HCC with cirrhosis group [34]. Qiao et al. (2017) had also reported that the N-glycosylation mutations on the MHR region accompanied with HBsAg-antiHBs co-existence was related to HCC [35]. Liu et al. (2013) further stated that the large N glycosylation of HBsAg antigen modulates HBsAg secretion, causes ER stress, expresses cell cycle and cell proliferation [36].

The mutant strains with amino acid changes at T or B cell epitopes on the PreS region can escape the immune surveillance that prolong the HBV infection. Mutants at specific regions of PreS/S genes may create premature stop codons, produce abnormal truncated proteins, disbalance the synthesis of surface proteins, result in retaining of HBV inside of the host cells, promote the endoplasmic reticulumn stress pathway, cause DNA oxidative damage and genome instability, upregulate cell cycles and lead to malignant transforming of hepatic cells [7, 25–27]. The PreS/S mutant strains enhance cell cycle progression through the down-regulating effects on the p53 and p21 pathways; upregulate the cyclin-dependent kinase 4, cyclin A, hamper HBsAg secretion, increase cellular proliferation [8].

Many other concerns related to the mutation strains and its replacements on virion secretory defect (at amino acid 172 on S gene) (Warner et al. [37]), on cell proliferation and transformation effects (at amino acids 95, 182, and 216 on S gene) (Huang et al. 2014 [38]) or predisposition of the HCC development (at amino acids 69, 95, 182, 216, 210 on S gene) [8, 38]. However, all of these concerned point-mutations were not found related to HCC in our study.

By study on liver tissue of HCC patients (2008), Hatazawa et al. had detected 2 PreS mutations (W4R and A60V) and more other PreS amino acid replacements at codon 5, 30, 35, 5, 54, 77, I84, 98, 102, 118, 123 and 124 [31]. Chen et al. (2008) had also reported W4P/R and other changes at codons 7, 81 on the PreS1, and at codon 68 on the S region related to HCC [39]. Several years later, the significantly higher frequencies of 3 PreS mutations at codons 4, 60 and 125 in HCC patients were recorded by Yin et al. (2010) [40], Zhang et al. [28]. Interestingly in a longitudinal study, Zhang had also observed the increasing of quasi-species complexity and diversity of the HBV strains during the progression to HCC; He had specially stated that the majority of these mutations existed at least 10 years in advance of development of HCC [41]. Zhang et al. (2017) had also repeatedly reported significantly higher rates of PreS deletion and other PreS mutations at codons 4, 27 and 167 in the HCC group [28].

Scientific reviewed on point-mutations that related to HCC, we realized that there were big differences on the patterns and characteristics of the amino acid changes related to HCC between studies. These differences might originate from the structure of study populations, HBV genotypes, the large spectrum of amino acid changes along the PreS1/PreS2/S sequences, and the interactions between mutations.

The multivariable analysis was applied twice in our study. Firstly, to adjust interactions between 19 point-mutations that had showed higher rates on the HCC group and recognized 6 mutations which had higher risks of HCC (Table 5). Secondly, to adjust for the confounding effects of personal and viral factors (Table 6). The final findings had recognized 5 mutations (W4P/R/Y on the PreS1 region and T47A/E/V/K, P120S/T, S174N, P203R on the S region) that significantly related to HCC. The findings that related to the first 3 mutations that were in agreement with other published papers, except the P203R which had not been well reported. Salpini et al. (2017) had stated that P203Q and the combination of P203R and S210R hampered the HBsAg secretion and increased cellular proliferation. The correlation of the C-terminus P203Q (17.4% vs 1.0%, p = 0.004), S210R (34.8% vs 3.8%, p<0.001) and of their combination with HCC had been reported in genotype A and D CHB patients [8].

Regarding to the OR values of mutations on the final multivariable analysis, 2 S mutations including 23 cases of T47A/E/V/K (S) and 21 cases of P120S/T (S) had revealed three folds increase in HCC risk associated with reasonable confidential intervals. On the contrary, three remaining mutations had only been detected on small numbers of cases with especially high ORs and wide 95% confidential intervals including 3 cases of W4P/R/Y (preS1), OR 11.56 (1.99–67.05); 4 cases of S174N (S), OR 29.73 (2.12–417.07); and 8 cases of P203R (S), OR 8.45 (1.43–50.06) (Table 6). A small sample size of this study resulted in a wider confidence interval with a larger margin of error for these sporadic mutations. It was suggested that a tighter confidence interval with values closer to the actual OR would be obtained if the sample size was increased. We had calculated the predictive values of these 5 point-mutations and had all found the high SPE and NPV values, but all revealed modest SEN due to small number of cases. S174N (S) for instance had been observed in our study with the highest OR and relative high PPV, NPV and specificity (75%, 81.1% and 99.5%, respectively) but its sensitivity was only 6.1% (S3 Table). If possible, the deep sequencing technique with its higher sensitivity could either potentially increase detection rates or improve the SEN values of these low frequency point-mutations. However, we were unable to perform it this time due to a large cost associated with the technique. Further studies are recommended in continuing upon findings of this study in which the direct sequencing would be the best and compulsory technique for better recognizing point mutations at quacispecies levels.

Contrarily, frequent amino acid replacements in our study were detected at the widely known structural and functional sequences such as N51Y/T/S/Q (30.4%), V68T/S/I (44.9%) on the S promoter; T/N87S/T/P (46.2%) on the HSP 70 (heat shock protein) and T125S/N/P (30.8%) on the NBS region. These structures are concerned by their role on the structure and morphology of HBV, the dual topology of L proteins (HSP70), the CAD—Cytosolic anchorage determinant), the virion morphogenesis (NBS—The nucleocapside binding site) and the S RNA transcription (The S promoter and the CCAAT/CBF) [42]. At the cellular level, the mutations at these functional regions has been known to contribute to the production and secretion of surface proteins, on the intracellular retention of envelope proteins and on the endoplasmic reticulum (ER) stress [43]. However, these above point-mutations had equally distributed on the HCC and non-HCC group in our study. More longitudinal cohorts need to be continued apart from this population because the diseases and HCC outcomes need at least one or more decades to appear.

There were some HCC related factors that were not included in the multivariable analysis such as Basal core promoter mutations, history of vaccination, HBsAg-antiHBs co-existence status, HBV genotype, cirrhotic status. Also, the combination of mutations and their interactive effects had not yet been analyzed. Other limitations of our study were also rooted from the study population that was not large enough for the low-rate mutations. A wide spectrum of significant mutations on the 3 regions (PreS1, PreS2 and S) and the interactive effects between mutations that need to be concretely clarified.

Further larger investigation and observation longitudinal studies were in need to be done to describe and analyse the relation between PreS/S mutation and HCC.

Conclusions

61% amino acid changes with a broad range of mutation rates were detected on the PreS1/PreS2/S regions of chronic HBV infected patients. The W4P/R/Y (on preS1 region) and T47A/E/V/K, P120S/T, S174N and P203R (on S region) were found related to HCC. Further investigation included cohort studies are recommended to continue to further investigate the relation of mutations on the HBV genome and HCC outcome.

Distributions of 6 mutations related to HCC in groups of personal and HBV characteristics.

(DOCX)

Click here for additional data file.

Distribution of personal characteristics and HBV viral markers in HCC and non HCC group (n = 247).

(DOCX)

Click here for additional data file.

Predictive values of point-mutations related to HCC (n = 247).

(DOCX)

Click here for additional data file.

(XLSX)

Click here for additional data file.

18 Nov 2021

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Additional Editor Comments:

4 independent reviewers together with editor reviewed this paper, all judged the importance of the findings obtained, however, the some major modifications are required especially, since S174N (S) point mutation showed the highest OR with significant P value, what are the sensitivity, specificity, PPV and NPV for this point mutation. It is also recommended to deep sequencing on some samples to see the rate of quasispecies of some of these significant mutations

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #3: Yes

Reviewer #4: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: My concerns regarding this manuscript are:

1-The history of HB vaccination and HBsAb status of the studied patients are not presented here, indeed the occurrence of Pre-S/S gene mutations may be affected by both factors.

2- The duration of HBV infection of included patients and its relation to pre-S/S mutations as well as the HCC occurrence should be shown and analyzed.

3- The method of liver fibrosis assessment is not mentioned in methodology, this should be clarified

4- It would be of clinical interest; to present the relation between liver inflammation status on one hand (e.g., ALT, AST or grade of liver inflammation on liver biopsy if available) and the pre-s/s mutations as well as HCC occurrence on the other hand

Reviewer #2: In the manuscript entitled "Mutations in the HBV PreS/S gene related to hepatocellular carcinoma in Vietnamese chronic HBV-infected patients", the authors performed a cross-sectional study including Vietnamese CHB-infected patients with high HBV-DNA >and performed PreS/S genes sequencing. The article is well presented despite the need for vigorous language revisions. My main concern is the value of generalising the presenting data which is limited to the Vietnamese population.

Reviewer #3: PONE-D-21-33925

The strong point of this manuscript

-Aim of the study is good and important

-good results

Weakness of the manuscript

1-Englsih editing is required

The follaoing is my comments

Abstract

Please add the following paragraph in new Materials section

Ine 35-35 page 8

247 CHB-infected patients were recruited, characterized by 68.8% males, 44.5%

HBV genotype C, 54.4% HBeAg positive, 23.1% fibrosis score ≥F3 and 19.8% HCC

Please add major hydrophilic region line 39 page 8 (with point-mutation on major hydrophilic region (MHR)

Please delete "We detected a wide spectrum of point-mutations on PreS/S regions in

Vietnamese CHB patients line" 48 page 8

Introduction

Please correct the reference Wang et al (2020)

Please correct the reference Dunford et al group (2012)

Materiasl and methods

Is ok

Statistics is included

Ethics Considerations is included

Results

The presentation of the results is more complicated and it is better if authors represented it with more simple figures

Discussion

Please delete the subtitles in the discussion section

There are several of references are not accrding to the style of the journal such as

(2017) [27], Choi et al (2019), [28]).

(Hudu el al 2015 [29], Kim et al 2018 [20], Hazawa 2018 [30],

Bui TTT et al (2017)

Kim et al (2018)

Qiao et. al (2017)

Liu et al (2013)

(Warner et al [34]),

(Huang 2014

(2008), Hatazawa et al

Chen et al (2008)

Yin et al (2010)

Please delete Recently, and correct the reference Zhang et al (2017)

References

Contains several recent references 2020, 2021

Reviewer #4: Huong, et al. investigated the correlation between preS/S of HBV genome mutations and HCC in HBV-Vietnamese patients. The study is interesting, however, there are some issues need to be addressed:

1- Is there any significant correlation between the mutations identified in the multivariable analysis in table 6 and sex, age and HBeAg negative.

2- Since S174N (S) point mutation showed the highest OR with significant P value, what are the sensitivity, specificity, PPV and NPV for this point mutation.

3- It is recommended to deep sequencing on some samples to see the rate of quasispecies of some of these significant mutations.

4- Tables 2,3 and 4 are very long, might move as supplementary tables.

5- In the abstract (at the end of third line of the results), it looks there are some information is missing here "and on PreS1 gene"

6- Any abbreviations should be spelled out in the first place, such as MHR in the abstract.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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16 Jan 2022

Response to reviewers

We would like to express our gratitude to the editors and reviewers of this manuscript for thorough review and providing us with highly constructive comments to improve it. We have revised the manuscript according to the suggestions and made appropriate changes where needed.

Editor's comments:

• Comments: We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

Reply: We removed the phrase “data not shown” in line 230, page 22, and replaced with data was shown completely in Table S2 "Distribution of personal characteristics and HBV viral markers in HCC and non HCC group". All relevant data are within the manuscript and its supporting information as File S2_Pres_S mutation related to HCC.xlxs.

• Comments: 4 independent reviewers together with editor reviewed this paper, all judged the importance of the findings obtained, however, the some major modifications are required especially, since S174N (S) point mutation showed the highest OR with significant P value, what are the sensitivity, specificity, PPV and NPV for this point mutation. It is also recommended to deep sequencing on some samples to see the rate of quasispecies of some of these significant mutations.

Reply: Regarding to the OR values of mutations on the final multivariable analysis, 2 S mutations (T47A/E/V/K(S) and P120S/T(S)) had revealed three fold increase in HCC risk and good confidential intervals. On the contrary, three remaining mutations had only been detected on small numbers of cases (W4P/R/Y(preS1) 3 cases, OR 11.56 (1.99-67.05); S174N(S) 4 cases, 29.73 (2.12-417.07); and P203R (S) 8 cases, OR 8.45 (1.43-50.06)) with especially high ORs (table 6). The wide 95% confidential intervals of OR with very high upper values of these intervals revealed that the sample size was not large enough for investigating these sporadic mutations. Ideally, the deep sequencing technique could potentially increase the detection rates due to its higher sensitivity. However, the cost effectiveness should be considered for the aims of studying these irregular mutations or investigating a larger population. We mentioned these explanations in line 350-360. Due to limited condition, we were unable to perform deep sequencing in this study to determine sensitivity, specificity, PPV and NPV for this point mutation S174N (S).

Reviewer #1 's opinions:

• Comments: The history of HB vaccination and HBsAb status of the studied patients are not presented here, indeed the occurrence of Pre-S/S gene mutations may be affected by both factors.

Reply: Thank you for your great feedback.

�  During sample collection, the history of HB vaccination was not recorded in 212 CHB patients whose serum samples had been stored during 2014-2016. It was only recorded in 35 CHB patients that were recruited in 2020-2021. Thus, there was no sufficient data available in all patients that can be utilized to analyze the relation of the HB vaccination to Pre-S/S gene mutations.

�  In line 294-299, we has clarified: "Moreover, among our study population, antiHBs that had been tested on 186 cases with clinical symptoms were tested antiHBs and had detected 37 cases (19.9%) with HBsAg-AntiHBs coexsistence, higher than the rates of 3-5% in other investigation. Therefore, it was presumed that CHB patients with varieties of presentations had been included in our study and contributed to the difference in rates of point-mutations compared to other studies".

• Comments: The duration of HBV infection of included patients and its relation to pre-S/S mutations as well as the HCC occurrence should be shown and analyzed.

Reply: Data from hospital electronic database cannot be used to directly respond to this question. Alternatively, the time from when patients had been diagnosed with CHB to the time of study participation was calculated using data from “year of CHB diagnosed” and “year of inclusion”.

• Comments: The method of liver fibrosis assessment is not mentioned in methodology, this should be clarified.

Reply: In line 117-123, it is added that "Cirrhosis was defined as having signs of portal hypertension (splenomegaly, ascites, vascular collaterals on abdominal wall, esophageal varices or portal hypertensive gastropathy on gastroscopy) and signs of insufficiency of liver function (palmar erythema, vascular spiders, low concentration of albumin (<35 g/dL), elevation of the international normalized ratio (INR >1.1), thrombocytopenia (<160,000/mm3)) as well as irregularity of hepatic surface on ultrasonography or F3 on Metavir score using Acoustic radiation force impulse (ARFI) measurement [22]". We based on the reference of "Udell JA, et al. Does this patient with liver disease have cirrhosis? JAMA. 2012;307(8):832-42".

• Comments: It would be of clinical interest; to present the relation between liver inflammation status on one hand (e.g., ALT, AST or grade of liver inflammation on liver biopsy if available) and the pre-s/s mutations as well as HCC occurrence on the other hand

Reply: In our data, we only used ALT cross-sectional data (one time at inclusion) and we didn't have information on liver biopsy. Relation between ALT (non repeated measurementy) and mutation and HCC were not in the scope of this study.

Reviewer #2 's opinions:

• Comments: My main concern is the value of generalising the presenting data which is limited to the Vietnamese population.

Reply: Thank you for your comment. With advantage of having decent number of patients in different stages of CHB enrolled in the study, our data can moderately represent the Vietnamese population. However, as the study was only conducted on patients with detectable HBV DNA, the rates of mutations are predicted to be possibly higher than general population. Therefore, in the scope of this study, we do not aim to generalize the frequencies of mutation. Instead, it is intended to determine the correlation between mutation of HBV on PreS/S genes and HCC in vietnamese population. Further studies are suggested to generalize data to chronic HBV infected HCC community.

Reviewer #3 opinions: Many thanks for such addition

• Comments: English editing is required

Reply: We edited academic English writing whole our manuscript.

• Comments: Please add the following paragraph in new Materials section

Iine 35-35 page 8--> 247 CHB-infected patients were recruited, characterized by 68.8% males, 44.5% HBV genotype C, 54.4% HBeAg positive, 23.1% fibrosis score ≥F3 and 19.8% HCC

Reply: It was summarized in the Result section.

• Comments: Please add major hydrophilic region line 39 page 8 (with point-mutation on major hydrophilic region (MHR) in abstract.

Reply: we edited “point-mutation on major hydrophilic region (MHR)” in abstract line 42.

• Comments: Please delete "We detected a wide spectrum of point-mutations on PreS/S regions in

Vietnamese CHB patients” line 48 page 8

Reply: We removed that sentence and replaced with " We detected 61% amino acid changes on PreS/S regions in Vietnamese CHB patients" line 50-51, page 2.

• Comments: There are several of references are not according to the style of the journal

Reply: we have completed a thorough review of all the references used in our study and made changes accordingly

• Comments: The presentation of the results is more complicated and it is better if authors represented it with more simple figures.

Reply: The tables are intended to describe the name of point-mutations along with their associated data for traceability purpose.

• Comments: In discussion: Please delete the subtitles in the discussion section, delete "Recently"

Reply: all subtitles and "Recently" are deleted as per comment.

Reviewer #4 's opinions:

• Comments: Is there any significant correlation between the mutations identified in the multivariable analysis in table 6 and sex, age and HBeAg negative.

Reply: We have completed the data analysis and revised the result line 221-224 and Table S1: The characteristics of these 6 point mutations had been analysed and found that W4P/R/Y (PreS1) (p=0.022) and T47A/E/V/K (S) (p<0.001) had significant higher rates on genotype C; P120S/T (S) had higher rates on genotype B (p<0.001), HBeAg (-) group (p=0.019) and low HBV DNA group (<5 log copies/mL) (p=0.013) (Table S1).

• Comments: Since S174N (S) point mutation showed the highest OR with significant P value, what are the sensitivity, specificity, PPV and NPV for this point mutation. It is recommended to deep sequencing on some samples to see the rate of quasispecies of some of these significant mutations (as above).

• Comments: Tables 2,3 and 4 are very long, might move as supplementary tables.

Reply: Tables are kept as it to support the clarification of data collected and considered critical to overall results.

• Comments: In the abstract (at the end of third line of the results), it looks there are some information is missing here "and on PreS1 gene"

Reply: We have added in line 38-40: The most common point mutations included N/H51Y/T/S/Q/P (30.4%), V68T (44.9%), T/N87S/T/P (46.2%) on PreS1 gene.

• Comments: Any abbreviations should be spelled out in the first place, such as MHR in the abstract.

Reply: "major hydrophilic region (MHR)" is added in line 42.

Sincerely,

Nguyen Thi Cam Huong

MD, PhD, Lecturer

Departments of Infectious diseases

University of Medicine and Pharmacy at Ho Chi Minh city

Submitted filename: Response to reviewers letter.docx

Click here for additional data file.

28 Feb 2022

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors thanks for your response , all comments have been addressed , I have no further comments regarding this work

Reviewer #3: The authors did all my revised comments, particularly in English editing, reference style, and deleting words recently from some sentences, and I accepted them in this form.

Reviewer #4: (No Response)

**********

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Reviewer #1: No

Reviewer #3: Yes: Ashraf A.Tabll

Reviewer #4: No

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6 Mar 2022

Response to reviewers

We would like to express our gratitude to the editors and reviewers for recognizing our effort to improve our manuscript. We have revised the manuscript according to the suggestions and made appropriate changes where needed.

Comments: "The manuscript has been significantly improved, however, the authors failed to comply with the reviewer comment regarding the calculation of PPV, NPV, sensitivity and specificity using deep sequencing or their available current data. This would give a clinical significance for their findings”.

Reply: It has been described in line 239-243 of the result section: "S174N (S) had the highest OR (29.73) with positive predictive value (PPV), negative predictive value (NPV), sentitivity (SEN), specificity (SPE) for predicting HCC respectively, 75%, 81.1%, 6.1%, 99.5%. The predictive values of all other 4 point-mutations related to HCC were concretely described in Table S3, based on our current available data."

We explained more in line 354-372 of discussion section: "Regarding to the OR values of mutations on the final multivariable analysis, 2 S mutations including 23 cases of T47A/E/V/K (S) and 21 cases of P120S/T (S) had revealed three folds increase in HCC risk associated with reasonable confidential intervals. On the contrary, three remaining mutations had only been detected on small numbers of cases with especially high ORs and wide 95% confidential intervals including 3 cases of W4P/R/Y (preS1), OR 11.56 (1.99-67.05); 4 cases of S174N (S), OR 29.73 (2.12-417.07); and 8 cases of P203R (S), OR 8.45 (1.43-50.06)) (table 6). A small sample size of this study resulted in a wider confidence interval with a larger margin of error for these sporadic mutations. It was suggested that a tighter confidence interval with values closer to the actual OR would be obtained if the sample size was increased. We had calculated the predictive values of these 5 point-mutations and had all found the high SPE and NPV values, but all revealed modest SEN due to small number of cases. S174N (S) for instance had been observed in our study with the highest OR and relative high PPV, NPV and specificity (75%, 81.1% and 99.5 %, respectively) but its sensitivity was only 6.1% (Table S3). If possible, the deep sequencing technique with its higher sensitivity could either potentially increase detection rates or improve the SEN values of these low-frenquency point-mutations. However, we were unable to perform it this time due to a large cost associated with the technique. Further studies are recommended in continuing upon findings of this study in which the direct sequencing would be the best and compulsory technique for better recognizing point mutations at quacispecies levels."

Sincerely,

Nguyen Thi Cam Huong

MD, PhD, Lecturer

Departments of Infectious diseases

University of Medicine and Pharmacy at Ho Chi Minh city, Vietnam

Submitted filename: Response to reviewers letter .docx

Click here for additional data file.

15 Mar 2022

Mutations in the HBV PreS/S gene related to hepatocellular carcinoma in Vietnamese chronic HBV-infected patients

PONE-D-21-33925R2

Dear Dr. Thi Cam Huong,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

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Additional Editor Comments (optional):

The Authors significantly improved the manuscript and efficiently answered reviewers' comments

Reviewers' comments:

22 Mar 2022

PONE-D-21-33925R2

Mutations in the HBV PreS/S gene related to hepatocellular carcinoma in Vietnamese chronic HBV-infected patients

Dear Dr. Thi Cam Huong:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Table 1

Characteristics of the study population (n = 247).

Characteristics		n (%)
Sex (male)		170 (68.8)
Age group ≥40		143 (57.9)
HBeAg positive		142 (57.5)
HBV DNA ≥5 (log10-copies/mL)		205 (83)
Genotype B (n = 245)		135 (54.7)
Fibrosis ≥F3		57 (23.1)
HCC		49 (19.8)